The positioning and dynamics of origins of replication in the budding yeast nucleus.

Heun P, Laroche T, Raghuraman MK, Gasser SM - J. Cell Biol. (2001)

Bottom Line:
We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase.If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic.This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo.

ABSTRACTWe have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)-tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

Figure 1: Three-dimensional reconstitution of confocal scans monitor nuclear integrity after combined IF/FISH labeling. (a–b) Double labeling of diploid budding yeast cells (GA-1190) by fluorescence in situ hybridization with a digoxigenin-derivatized subtelomeric repeat probe (Y′, green) and nuclear pore immunostaining (Mab414, red). (a) Cells synchronized at the G1/S border were fixed in the absence of detergents before processing for FISH. They remain structurally intact as judged by the presence of three to eight clusters of the subtelomeric Y′ element signals and their peripheral localization. Confocal sections were taken along the z axis of the nucleus and were reconstituted and deconvolved in 3-D (Imaris®). Three different views of the reconstituted nucleus are shown in the lower part of each panel, corresponding to the x–y, x–z, and y–z plane. (b) Cells were fixed in the presence of 0.1% Triton X-100 (see Materials and Methods). The nuclei are flattened (compare x–z with y–z planes) as nuclear organization is gradually destroyed. (c) Cells were fixed after exposure to 0.1% Triton X-100, a technique called nuclear spreading (Klein et al. 1992). In the samples shown in c, nuclear pore staining is routinely lost, probably due to solubilization of the pore protein. In this case, to visualize the nucleus, DNA was stained with POPO-3 (shown in red). (d and e) Double staining of cells that were fixed as in a. In this case, the DNA is visualized with YOYO-3 (green) and the nuclear pore is red (Mab414, see above). (f) A line profile histogram shows the DNA distribution along a line for the nucleus shown in e combined with the nuclear pore staining. a–c were collected on an LSM 410 confocal microscope and d–e on an LSM 510 confocal microscope (Carl Zeiss, Inc.). Scale bars: 2 μm.

Mentions:
Cells were prepared for immunofluorescence and FISH following the standard fixation method described in Gotta et al. 1999, except that cells were fixed in growth medium for 10 min at 30°C in 4% paraformaldehyde before spheroplasting. Fixed cells were washed three times and the conversion to spheroplasts was done with 300 U/ml of lyticase and 0.6–1.2 mg/ml of Zymolyase (20T) for ∼15 min. The arrest of cdc4-3 and cdc7-1 strains at the restrictive temperature results in more fragile cell walls and requires use of less lyticase. For alternative preparations (see Fig. 1), cells were fixed in the presence of 0.1% Triton X-100 or fixed after exposure to detergent as described (Klein et al. 1992). To visualize DNA, we use POPO-3, YOYO-3, or TOTO-3 (Molecular Probes) as indicated.

Figure 1: Three-dimensional reconstitution of confocal scans monitor nuclear integrity after combined IF/FISH labeling. (a–b) Double labeling of diploid budding yeast cells (GA-1190) by fluorescence in situ hybridization with a digoxigenin-derivatized subtelomeric repeat probe (Y′, green) and nuclear pore immunostaining (Mab414, red). (a) Cells synchronized at the G1/S border were fixed in the absence of detergents before processing for FISH. They remain structurally intact as judged by the presence of three to eight clusters of the subtelomeric Y′ element signals and their peripheral localization. Confocal sections were taken along the z axis of the nucleus and were reconstituted and deconvolved in 3-D (Imaris®). Three different views of the reconstituted nucleus are shown in the lower part of each panel, corresponding to the x–y, x–z, and y–z plane. (b) Cells were fixed in the presence of 0.1% Triton X-100 (see Materials and Methods). The nuclei are flattened (compare x–z with y–z planes) as nuclear organization is gradually destroyed. (c) Cells were fixed after exposure to 0.1% Triton X-100, a technique called nuclear spreading (Klein et al. 1992). In the samples shown in c, nuclear pore staining is routinely lost, probably due to solubilization of the pore protein. In this case, to visualize the nucleus, DNA was stained with POPO-3 (shown in red). (d and e) Double staining of cells that were fixed as in a. In this case, the DNA is visualized with YOYO-3 (green) and the nuclear pore is red (Mab414, see above). (f) A line profile histogram shows the DNA distribution along a line for the nucleus shown in e combined with the nuclear pore staining. a–c were collected on an LSM 410 confocal microscope and d–e on an LSM 510 confocal microscope (Carl Zeiss, Inc.). Scale bars: 2 μm.

Mentions:
Cells were prepared for immunofluorescence and FISH following the standard fixation method described in Gotta et al. 1999, except that cells were fixed in growth medium for 10 min at 30°C in 4% paraformaldehyde before spheroplasting. Fixed cells were washed three times and the conversion to spheroplasts was done with 300 U/ml of lyticase and 0.6–1.2 mg/ml of Zymolyase (20T) for ∼15 min. The arrest of cdc4-3 and cdc7-1 strains at the restrictive temperature results in more fragile cell walls and requires use of less lyticase. For alternative preparations (see Fig. 1), cells were fixed in the presence of 0.1% Triton X-100 or fixed after exposure to detergent as described (Klein et al. 1992). To visualize DNA, we use POPO-3, YOYO-3, or TOTO-3 (Molecular Probes) as indicated.

Bottom Line:
We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase.If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic.This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo.

ABSTRACTWe have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)-tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.